(i). Field of the Invention
The present invention relates to tissue cultures of plant cells, particularly monocotyledonous plant cells, quite particularly corn, rice, wheat and barley cells, and improved techniques to obtain genetically transformed plant cells and plants.
(ii). Description of the Related Art
Over the years many techniques for the genetic transformation of plants have been developed. These methods have as their ultimate goal the obtaining of a transgenic plant, in which all cells contain a foreign DNA comprising a gene of interest (the so-called transgene) stably integrated in their genome, particularly their nuclear genome.
Transformation is a complex process which always involves the contacting of starting cells with a DNA, usually a DNA comprising foreign gene(s) of interest. The contacting of the cells with the DNA is carried out under conditions that promote the uptake of the DNA by the cells and the integration of the DNA, including the gene(s) of interest into the genome of the cell.
Starting cells for transformation are usually cells that have been cultured in vitro for some time. After contacting the cells with the DNA, the transformed cells generally need to be cultured in vitro for a certain period in order to separate the transformed cells from the non-transformed cells and to regenerate transformed plants from the transformed cells.
Different plant transformation methods have been described and can be classified into direct DNA transfer methods (e.g. electroporation, PEG-mediated DNA uptake, biolistics) or Agrobacterium-mediated DNA transfer. Vasil (1994) and Christou (1994) have reviewed available plant transformation methods for cereals. Agrobacterium-mediated DNA transfer is one of the most efficient means of DNA transfer into plant cells, and requires probably the least technological hardware of the different transformation methods. Also quantitatively, the transformed plants obtained by Agrobacterium-mediated DNA transfer are superior, in comprising a smaller number of transgenes inserted at different positions in the chromosome, and in that aberrant transgenes have a lower occurrence. Agrobacterium-mediated DNA transformation of plants is based on the capacity of certain Agrobacterium strains to introduce a part of their Ti-plasmid, i.e. the T-DNA, into plant cells and to integrate this T-DNA into the nuclear genome of the cells. It was found that the part of the Ti-plasmid that is transferred and integrated is delineated by specific DNA sequences, the so-called left and right T-DNA border sequences and that the natural T-DNA sequences between these border sequences can be replaced by foreign DNA (European Patent Publication "EP" 116718; Deblaere et al., 1987).
Agrobacterium-mediated transformation of monocotyledonous plants has been reported several times (see infra). The applicability of the reported methods has been limited however, to specific species or genotypes, or required the use of specific tissues, or specialized Agrobacterium strains. For most of the reported methods, transformation efficiency can still largely be improved.
Hooykaas-Van Slogteren et al. (1984), describes the detection of Ti-plasmid gene expression in two monocot species (Chlorophytum capense and Narcissus cv `Paperwhite`) infected with tumorogenic Agrobacterium strains.
Hernalsteens et al. (1984) and Bytebier et al. (1987), describe transformation of Asparagus officinalis using natural Agrobacterium tumefaciens isolates, as well as Agrobacterium tumefaciens strains comprising a non-oncogenic T-DNA.
U.S. Pat. No. 5,164,310 describes a method to transform plants (comprising corn and wheat) by inoculating excised and cultured shoot apices of the plants with Agrobacterium tumefaciens.
U.S. Pat. Nos. 5,187,073 and 5,177,010 describe a method of producing transformed Graminaea (corn) comprising making a wound in a seedling in an area of the seedling containing rapidly dividing cells and inoculating the wound with vir+ Agrobacterium tumefaciens.
PCT patent publication WO 92/09696 describes the use of compact embryogenic callus (i.e. Type I callus in corn) and immature embryos (wounded either mechanically or enzymatically) of monocotyledonous plants (e.g. corn and rice) as starting material for transformation procedures.
EP 0604662 A1 describes a method of transforming cultured tissues of a monocotyledon under or after dedifferentiation with a bacterium of the genus Agrobacterium containing desired genes. EP 0672752 A1 describes a method of transforming a scutulum of a non-dedifferentiated immature embryo of a monocotyledon with an Agrobacterium. Both applications describe the use of Agrobacterium strains having a plasmid containing a DNA fragment originating from the virulence region of Ti-plasmid pTiBo542 in addition to the Ti or Ri plasmid.
Raineri et al. (1990) describes transformation of embryo-derived cultures of two rice cultivars, wounded in the scutellar region, using an Agrobacterium mediated gene transfer system.
Chan et al. (1993) describes a method to transform immature embryos of rice that have been cultured for 2 days in the presence of 2,4-dichlorophenoxy acetic acid ("2,4-D") by inoculation with Agrobacterium strains on a medium containing potato suspension culture cells.
Mooney et al., (1991) describes a method for Agrobacterium-mediated introduction of a kanamycin-resistant gene into enzyme-treated embryos of wheat.
Induction of the vir genes of Ti plasmids or helper plasmids of Agrobacteria strains by incubation of the bacteria with acetosyringone prior to cocultivation to enhance transformation, and addition of acetosyringone during cocultivation of the plant cells with the bacteria has been reported (Van Wordragen and Dons, 1992; Jacq et al., 1993; James et al., 1993).
Guivarc'h et al. (1993) describes improvement of transient Agrobacterium-mediated transformation of carrot root discs by a short pretreatment of these discs for ten minutes with acetosyringone.